The location of fluorescence probes with charged groups in model membranes

The location of commonly used charged fluorescent membrane probes in membranes was determined in order to: (1) investigate the relationship between the structure of hydrophobic molecules and their depth within membranes; and (2) aid interpretation of experiments in which these fluorescent probes are used to examine membrane structure. Membrane depth was calculated using parallax analysis, a method in which the quenching induced by lipids carrying a nitroxide group at different locations in the membrane is compared. Shallow locations were found for xanthene dyes (fluorescein, eosin, Texas Red and rhodamine) both in free form and when attached either to the headgroup of phospholipids or long hydrocarbon chains. The exact structure of the xanthene and the nature of its linkage to lipid had only a modest effect on membrane location, which ranged between 19 and 24 A from the center of the bilayer in a charged state. Thus, the location of these fluorophores largely reflects their intrinsic properties rather than the nature of the groups to which they are attached. Furthermore, cationic and anionic xanthene derivatives had similar depths, indicating the type of charge does not have a large effect on depth. Consistent with this conclusion, shallow locations were also found for other hydrocarbon chain-linked cationic (acridine orange and styrylpyridinium) and anionic (coumarin, anilinonaphthalenesulfonic acid (ANS), and toluidinylnaphthalenesulfonic acid (TNS)) charged probes. These all located at 16-18 A from the bilayer center. We conclude that both anionic and cationic molecules that are otherwise hydrophobic predominantly occupy shallow locations within the polar headgroup region of the bilayer no matter how hydrophobic the molecule to which they are linked. This depth is significantly shallower than that occupied by most previously studied uncharged polar molecules that locate near the membrane surface. Consistent with this conclusion, a 2-4 A deeper location was found for xanthene probes with no net charge. In other experiments, methods to avoid chemical reactions that can distort the measurement of depth by fluorescence quenching were developed.